Application and determination method for promoting stable carbon deposition of soil based on microbial metrology metabolism

Abstract

The invention belongs to the technical field of carbon circulation, and particularly discloses an application for promoting stable carbon deposition of soil based on microbial metered metabolism, straw carbon, nitrogen, phosphorus and sulfur are added, and the stoichiometric ratio of C to N to P to S added into the soil is adjusted to 10000: (1034-2004): (167-494): (133-264). Therefore, the C utilization efficiency of microorganisms is improved, and conversion and accumulation of straw carbon into soil organic carbon are accelerated. Under the NPS3 provided by the invention, more bacteria can be observed to be mobilized, so that the carbon digestion and conversion of the plant-derived residues and the carbon burying and accumulation of the microorganism-derived residues are accelerated. The invention also provides a determination method for promoting soil stable carbon deposition based on microbial metered metabolism, by setting a control group and a treatment group, the palatability of a metabolic substrate to microorganisms, especially the palatability of bacteria, is improved, and the effect of accelerating the conversion of straw carbon to soil organic carbon is verified.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon cycle, and specifically relates to a determination method and application of promoting stable soil carbon deposition based on microbial metabolic measurement. Background Art

[0002] The carbon cycle is an important part of the terrestrial ecological cycle system. For example, Chinese patent application No. 202110564048.5 discloses a method for returning straw to the field and applying inorganic nutrients to increase the organic carbon content in the soil. This method believes that the difference in elemental composition between straw and soil limits the efficient humification process of straw carbon. Therefore, this method adds inorganic nutrients while returning the straw to the field, and follows the nutrient requirement ratio of C:N:P:S=10000:860:169:129 in the straw C conversion. The addition of nutrients and exogenous organic materials will enhance the mineralization and decomposition of natural SOC. At the same time, straw carbon will form new soil SOC at a constant ratio of carbon to nutrients. That is, the principle is to make the ratio of straw C to inorganic nutrients close to the stoichiometric ratio in soil stable organic matter through the addition of inorganic nutrients.

[0003] However, after returning straw to the fields using the above method, straw decomposition is still slow, and the carbon source humification efficiency does not meet expectations, which limits the process and intensity of straw carbon transfer into the soil carbon pool. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a determination method and application of promoting stable soil carbon deposition based on microbial metabolic metabolism, which improves the palatability of metabolic substrates to microorganisms, especially bacteria, by adjusting the ratio of C, N, P, and S in the soil, thereby improving the utilization efficiency of C by microorganisms and accelerating the conversion and stabilization of straw carbon into soil organic carbon.

[0005] The specific technical solution adopted by the present invention is: Based on the application method of promoting stable soil carbon accumulation by microbial stoichiometric metabolism, the stoichiometric ratio of C:N:P:S in the soil substrate was adjusted to 10000:(1034-2004):(167-494):(133-264) by adding straw carbon and nutrients nitrogen, phosphorus and sulfur.

[0006] The ideal stoichiometric ratio of C:N:P:S is 10000:2004:494:264.

[0007] The determination method based on microbial metabolic promotion of soil stable carbon accumulation includes the following steps: S1. Establish a control group NPS0, and treatment groups NPS1-NPS3 with combined addition of straw carbon and nutrients nitrogen, phosphorus and sulfur. The stoichiometric ratio of C:N:P:S added to the soil in each treatment of NPS1-NPS3 was adjusted to 10000:(1034-2004):(167-494):(133-264); S2. Soil substrate samples were collected from the topsoil and bottomsoil, air-dried, sieved, and impurities removed, and placed in mesh bags according to the four treatment groups NPS0-NPS3. Nine replicates were maintained for each treatment of the topsoil and bottomsoil; S3. Three uniform and regular soil pits were dug in the field as landfill areas. Three replicates of the four treatments of topsoil and bottomsoil were buried in each pit at different depths of topsoil and bottomsoil, and the corresponding layers of soil were used for backfilling; S4. Sow corn in the landfill area and regularly take duplicate samples of each treatment from the landfill area to determine the content of microbial residue carbon and plant residue carbon.

[0008] The treatment group NPS0 was the traditional straw return method, and the amount of nitrogen, phosphorus, and sulfur added to NPS0 was 0, which served as the control group; The C:N:P:S stoichiometric ratio of NPS1 is 10000:1034:167:133 to match the metabolic requirements of fungi; The metabolic demand matched by the C:N:P:S stoichiometric ratio of NPS2 is between that of fungi and bacteria, with a stoichiometric ratio of 10000:1556:238:171; The C:N:P:S stoichiometric ratio of NPS3 is 10000:2004:494:264 to match the metabolic requirements of bacteria.

[0009] In step S1, the C added incrementally in the treatment groups NPS0-NPS3 is straw carbon, which is obtained by crushing straw into 2-5 mm fragments, adding 2 g of fragments to every 100 g of soil substrate sample after air drying, sieving and impurity removal, and the concentrations of C, N, P and S in the fragments are 41.76%, 0.974%, 0.120% and 0.119%, respectively; the stoichiometric ratio of the substrate is adjusted by adding nutrient solutions NS1, NS2 or NS3 containing ammonium nitrate, potassium dihydrogen phosphate and ammonium sulfate to the treatment groups NPS1-NPS3, and no nutrient solution is added to the treatment group NPS0, and the concentrations of N, P and S in NS1 are 6.421, 1.791 and 0.952 g L −1 , and the concentrations in NS2 were 19.502, 3.521, and 1.902 g L −1 , and the concentrations in NS3 were 30.721, 9.981, and 4.232 g L −1, the amount of nutrient solution NS1, NS2, and NS3 added was 1 ml per 100 g of soil sample.

[0010] The topsoil is soil with a depth of 0-20 cm, and the bottomsoil is soil with a depth of 20-40 cm. Before the soil substrate samples are treated with the treatment groups NPS0-NPS3, the soil moisture content of the soil substrate samples is adjusted to 60% of the field water holding capacity with distilled water. The mesh bag used is a nylon mesh bag with a mesh bag pore size of 0.048 mm.

[0011] The pit dimensions are 1.5 m long, 0.5 m wide, 0.4 m deep, and 0.6 m apart. Each treatment is spaced 20 cm apart. The bottom layer of each treatment of the bottom soil is placed at a depth of 20-40 cm, and the top soil is placed at a depth of 0-20 cm.

[0012] The corn was planted with a spacing of 27 cm between plants and 60 cm between rows. All repeated samples from a landfill area were collected for measurement at 30, 90 and 150 days after sowing.

[0013] The determination method of the microbial residue carbon is as follows: a. Glucosamine GlcN and muramic acid MurA were evaluated as biomarkers. One gram of air-dried sample was hydrolyzed in a hydrolysis tube at 105°C for 8 hours. b. Add 100 μL of inositol to the tube, mix, filter and spin dry at 65°C; c. The residue was dissolved in 20 mL of pure water, centrifuged, and freeze-dried. The freeze-dried residue was dissolved in 5 mL of anhydrous methanol, centrifuged, transferred to a 5 mL derivatization bottle, and dried with N2 gas; d. Add 1 mL of pure water and 100 μL of N-methylglucosamine and freeze-dry; e. Prepare three standard samples, add 100 μL of mixed standard solution to each sample and freeze-dry; f. The gas-dried samples and standard samples were sealed with 300 μL of derivatization reagent and shaken for 30 seconds; g. In a water bath, add 1 mL of acetic anhydride, 1.5 mL of dichloromethane and 1 mL of hydrochloric acid to each sample in sequence; h. After standing and stratification, the inorganic phase was removed and the above operation was repeated 3 times; the remaining organic phase was dried with N2 gas at 45 ° C; the residue was dissolved with 300 μL of diluent and detected by gas chromatography; i. Calculate bacterial residue carbon BNC and fungal residue carbon FNC according to formulas (1) and (2): BNC = C MurA × 45 (1) FNC = (CGlcN / 179.17 – 2 × C MurA / 251.23) × 179.17 × 9 (2) Among them, C MurA is the concentration of MurA (mg g –1 ), 45 is the conversion coefficient between MurA and BNC, C GlcN is the concentration of GlcN (mg g –1 ), 179.17 and 251.23 are the relative molecular weights of GlcN and MurA, respectively, and 9 is the conversion coefficient between GlcN and FNC; Microbial residue carbon (MNC) was estimated as the sum of FNC and BNC.

[0014] The determination method of the plant residue carbon is as follows: A. Determination of PNC of plant residue carbon Ligninphenol was used as a biomarker. Ligninphenol includes vanillylphenol V, eugenol S and cinnamylphenol C. 0.5 g of air-dried sample was placed in a Monad reactor, and 500 mg of copper oxide, 100 mg of ammonium sulfate, 50 mg of glucose, 0.4 ml of internal standard solution and 15 ml of 2 M sodium hydroxide were added; B. The reactor was oxidized at 150°C for 150 minutes; C. Centrifuge the oxidation product, transfer the supernatant to another centrifuge tube and store it in the dark for 1 hour. Centrifuge again, transfer the supernatant to a volumetric flask, titrate to 100 ml with ethyl acetate solution, and evaporate in a rotary evaporator. Dissolve the residue with 0.5 ml of internal standard solution, transfer to a 3 ml derivatization bottle, and dry with N2 gas; the internal standard solution is prepared by dissolving 10 mg of ethyl vanillin in 100 ml of 2 M sodium hydroxide solution. D. The dried residue was dissolved with 50 μl pyridine and 100 μl N,O-bis(trimethylsilyl)trifluoroacetamide, derivatized at 60°C for 10 min, and detected by gas chromatography; The PNC of plant residue carbon is calculated according to formula (3): PNC = (C V / 0.33 + C S / 0.9 + C C ) / (0.08 × 1000) (3) Among them, C V , C S and C C are the carbon concentrations in vanillylphenol V, eugenol S, and cinnamylphenol C (ug g –1 ), the coefficients of CuO oxidation to release V, S and C are 0.33, 0.90 and 1 respectively; The minimum lignin concentration in major crop plant residues was set at 0.08; 1000 is the conversion factor between ug and mg.

[0015] The beneficial effects of the present invention are: The present invention enhances the palatability of microorganisms, especially bacteria, by adding straw carbon and nitrogen, phosphorus and sulfur to the soil, thereby improving the conversion and sequestration efficiency of C by microorganisms and accelerating the stable accumulation of straw carbon in the soil organic carbon pool. Under the NPS3 of the present invention, more bacteria can be observed to be mobilized, thereby accelerating the digestion and conversion of plant-derived residual carbon and the accumulation of microbial-derived residual carbon. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Evolution of microbial residue carbon and plant residue carbon in surface and deep soils under different substrate stoichiometries during the whole maize growing season.

[0017] Different lowercase or uppercase letters above the bars indicate significant differences (P < 0.05) between NPS0–NPS3 in the surface and deep soil layers, respectively. Asterisks indicate significant differences (P < 0.05) between the surface and subsurface layers.

[0018] CK, soil only; NPS0, soil with straw only; NPS1–NPS3, soil with added straw and increasing nutrients.

[0019] Figure 2 .Evolution of fungal and bacterial residue carbon in surface and deep soils over time under different substrate stoichiometries.

[0020] Different lowercase or uppercase letters above the bars indicate significant differences (P < 0.05) between NPS0–NPS3 in the surface and deep soil layers, respectively. Asterisks indicate significant differences (P < 0.05) between the surface and subsurface layers.

[0021] CK, soil only; NPS0, soil with straw only; NPS1–NPS3, soil with added straw and increasing nutrients.

[0022] Figure 3 .The ratio of cinnamyl alcohol-vanillylphenol (C / V) to eugenylphenol-vanillylphenol (S / V) as a function of time at different substrate stoichiometric ratios.

[0023] The red and blue dashed lines represent the mean values ​​of the surface layer and subsurface layer, respectively (n = 3). Asterisks indicate significant differences between the surface layer and subsurface layer (P < 0.05).

[0024] CK, soil only; NPS0, soil with straw only; NPS1–NPS3, soil with added straw and increasing nutrients.

[0025] Figure 4 . Temporal changes in lignin phenols (vanillylphenol V, eugenol S, and cinnamylphenol C) in the top and bottom soils under different substrate stoichiometric ratios.

[0026] Different lowercase or uppercase letters above the bars indicate significant differences (P < 0.05) between NPS0 to NPS3 in the top soil and bottom soil.

[0027] CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added.

[0028] Figure 5 .Activities of enzymes for carbon (a, b, c), nitrogen (d, e, f) and phosphorus (g, h, i) extraction at three sampling times under different nutrient addition levels.

[0029] Different lowercase or uppercase letters above the bars indicate significant differences (P < 0.05) between NPS0–NPS3 in the surface and deep soil layers, respectively. Asterisks indicate significant differences (P < 0.05) between the surface and subsurface layers.

[0030] BG, β-glucosidase; LAP, L-leucine aminopeptidase; AP, acid phosphatase; CK, soil only; NPS0, soil with straw only; NPS1–NPS3, soil with straw and increasing nutrients.

[0031] Figure 6 . Composition of bacterial communities in surface and deep soil layers under different substrate stoichiometry at days 30 and 150.

[0032] Lowercase letters next to the error bars indicate significant differences between NPS0–NPS3 (P < 0.05).

[0033] CK, soil only; NPS0, soil with straw only; NPS1–NPS3, soil with added straw and increasing nutrients.

[0034] Figure 7 Relative effects of soil variables on microbial residue C (MNC) and plant residue C (PNC) in surface and deep soil layers under oligotrophic (a, b) and eutrophic conditions (c, d), expressed as the percentage increase in mean squared error (MSE).

[0035] Significant variables are marked in green and red in the surface layer and subsurface layer, respectively (P < 0.05).

[0036] SM, soil moisture; AN, soil available nitrogen; SAP, soil available phosphorus; BG, β-glucosidase; AP, acid phosphatase; EVtopsoil / subsoil, total explained variance in the surface or subsoil; NPS1, soil with straw and nutrients matched to fungal requirements; NPS3, soil with straw and nutrients matched to bacterial requirements.

[0037] Figure 8 Schematic representation of MNC and PNC formation in surface and deep soils under conditions of substrate stoichiometry matching (from oligotrophic to eutrophic).

[0038] MNC, microbial residue carbon; PNC, plant residue carbon; FNC, fungal residue carbon; BNC, bacterial residue carbon; V, S and C, vanillylphenol, eugenol and cinnamylphenol; Nu, nutrient availability; SM, soil moisture; AN, soil available nitrogen; SAP, soil available phosphorus; Ace-B / F, Ace index of bacteria / fungi; Pla, Planktonic bacteria; Bac, Bacteroidetes; Gem, Gemmatimonadetes; Act, Actinobacteria; Aci, Acidobacteria; Asc, Ascomycota; Mor, Mortierella; Glo, Glomus; BG, β-glucosidase; LAP, L-leucine aminopeptidase; AP, acid phosphatase; P, soil phosphorus availability.

[0039] Fig. 9 . Soil moisture content in the top and bottom soils at different substrate stoichiometric ratios during the maize growing season at days 30, 90, and 150.

[0040] Different lowercase or uppercase letters indicate significant differences (P < 0.05) between NPS0 to NPS3 in the top and bottom soils. Asterisks indicate significant differences (P < 0.05) between the top and bottom soils.

[0041] CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added.

[0042] Fig.10 . Cellobiohydrolase (CBH) and β-N-acetylglucosaminidase (NAG) activities at three sampling time points under different nutrient addition levels in the top and bottom soils.

[0043] Asterisks indicate significant differences between the top and bottom soils (P < 0.05).

[0044] CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added.

[0045] Fig.11 . Alpha diversity of bacteria and fungi in top and bottom soils at three sampling time points under different nutrient addition levels during the maize growing season.

[0046] Different lowercase or uppercase letters indicate significant differences (P < 0.05) between NPS0 to NPS3 in the top and bottom soils. Asterisks indicate significant differences (P < 0.05) between the top and bottom soils.

[0047] CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added.

[0048] Fig.12 Fungal community composition (at the phylum level) in the top and bottom soils at days 30 and 150 under different substrate stoichiometric ratios.

[0049] Different lowercase letters above the error bars indicate significant differences between NPS0 and NPS3 (P < 0.05).

[0050] CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments: Specific embodiment 1, the present invention discloses an application method for promoting stable soil carbon deposition based on microbial stoichiometric metabolism, by adding straw carbon and nitrogen, phosphorus and sulfur, the stoichiometric ratio of C:N:P:S added to the soil is adjusted to 10000:(1034-2004):(167-494):(133-264). As shown in Table 1 below; Table 1: Substrate addition amount and its stoichiometric ratio CK is soil only, as a large reference; NPS0, soil plus straw, as a control group; NPS1–NPS3, soil plus straw and incremental nutrient addition. In the table, "Straw carbon" is the ideal humus content of straw carbon; C:N:P:S, the stoichiometric ratio of carbon, nitrogen, phosphorus and sulfur in the substrate. The "+" in the data indicates the amount of additional nutrients added to each treatment.

[0052] Preferably, the stoichiometric ratio of C:N:P:S is 10000:2004:494:264.

[0053] The determination method based on microbial metabolic promotion of soil stable carbon accumulation includes the following steps: S1. Establish NPS0 as the control group and treatment groups NPS1-NPS3 and add straw carbon and nitrogen, phosphorus and sulfur. The stoichiometric ratio of C:N:P:S added to the soil in each treatment of NPS1-NPS3 was adjusted to 10000:(1034-2004):(167-494):(133-264); S2. Soil substrate samples were collected from the topsoil and bottom soil, air-dried, sieved and impurities removed, and placed in mesh bags according to the four treatment groups NPS0-NPS3. Nine replicates were maintained for each treatment of the topsoil and bottom soil; S3. Three pits were dug in the field as landfill areas. Three replicates of the four treatments were buried in each pit at different depths of the topsoil and bottomsoil, and the corresponding layers of soil were used for backfilling; S4. Sow corn in the landfill area and regularly take duplicate samples of each treatment from the landfill area to determine the content of microbial residue carbon and plant residue carbon.

[0054] The treatment group NPS0 was the traditional straw return method, and the amount of nitrogen, phosphorus, and sulfur added to NPS0 was 0, which served as the control group; The C:N:P:S stoichiometric ratio of NPS1 is 10000:1034:167:133 to match the metabolic requirements of fungi; The metabolic demand matched by the C:N:P:S stoichiometric ratio of NPS2 is between that of fungi and bacteria, with a stoichiometric ratio of 10000:1556:238:171; The C:N:P:S stoichiometric ratio of NPS3 is 10000:2004:494:264 to match the metabolic requirements of bacteria.

[0055] In step S1, the C added incrementally in the treatment groups NPS0-NPS3 is straw carbon, which is obtained by crushing straw into 2-5 mm fragments, adding 2 g of the fragments to every 100 g of soil substrate sample after air drying, sieving and impurity removal, and the concentrations of C, N, P and S in the fragments are 41.76%, 0.974%, 0.120% and 0.119%, respectively; the stoichiometric ratio of the substrate is adjusted by adding nutrient solutions NS1, NS2 or NS3 containing ammonium nitrate, potassium dihydrogen phosphate and ammonium sulfate to the treatment groups NPS1-NPS3, and no nutrient solution is added to the treatment group NPS0, and the concentrations of N, P and S in NS1 are 6.421, 1.791 and 0.952 g L −1 , and the concentrations in NS2 were 19.502, 3.521, and 1.902 g L −1 , and the concentrations in NS3 were 30.721, 9.981, and 4.232 g L −1, the amount of nutrient solution NS1, NS2, and NS3 added was 1 ml per 100 g of soil substrate sample.

[0056] The topsoil is soil with a depth of 0-20 cm, and the bottomsoil is soil with a depth of 20-40 cm. Before the soil substrate samples are treated with the treatment groups NPS0-NPS3, the soil moisture content of the soil substrate samples is adjusted to 60% of the field water holding capacity with distilled water. The mesh bag used is a nylon mesh bag with an aperture of 0.03-0.05 mm.

[0057] The pit dimensions are 1.5 m long, 0.5 m wide, 0.4 m deep, and 0.6 m apart. Each treatment is spaced 20 cm apart. The placement depth of each treatment in the bottom soil is 20-40 cm, and the placement depth of each treatment in the top soil is 0-20 cm.

[0058] The corn was planted with a spacing of 27 cm between plants and 60 cm between rows. All repeated samples from a landfill area were collected for measurement at 30, 90 and 150 days after sowing.

[0059] The determination method of the microbial residue carbon is as follows: a. Glucosamine GlcN and muramic acid MurA were evaluated as biomarkers. One gram of air-dried sample was hydrolyzed in a hydrolysis tube at 105°C for 8 hours. b. Add 100 μL of inositol to the tube, mix, filter and spin dry at 65°C; c. The residue was dissolved in 20 mL of pure water (pH 6.6-6.8), centrifuged (4000 rpm, 10 minutes), and freeze-dried. The freeze-dried residue was dissolved in 5 mL of anhydrous methanol, centrifuged, transferred to a 5 mL derivatization bottle, and dried with N2 gas; d. Add 1 mL of pure water and 100 μL of N-methylglucosamine and freeze-dry; e. Prepare three standard samples, add 100 μL of mixed standard solution to each sample and freeze-dry; f. The gas-dried samples and standard samples were sealed with 300 μL of derivatization reagent and shaken for 30 seconds; g. In a water bath, add 1 mL of acetic anhydride, 1.5 mL of dichloromethane and 1 mL of hydrochloric acid to each sample in sequence, wherein the concentration of hydrochloric acid is 1 M; h. After standing and stratification, the inorganic phase was removed and the above operation was repeated 3 times; the remaining organic phase was dried with N2 gas at 45°C; the residue was dissolved with 300 μL of diluent and detected by gas chromatography, and the diluent was ethyl acetate: ethane in a volume ratio of 1:1; i. Calculate bacterial residue carbon BNC and fungal residue carbon FNC according to formulas (1) and (2): BNC = C MurA × 45 (1) FNC = (C GlcN / 179.17 – 2 × C MurA / 251.23) × 179.17 × 9 (2) Among them, C MurA is the concentration of MurA (mg g –1 ), 45 is the conversion coefficient between MurA and BNC, C GlcN is the concentration of GlcN (mg g –1 ), 179.17 and 251.23 are the relative molecular weights of GlcN and MurA, respectively, and 9 is the conversion coefficient between GlcN and FNC; Microbial residue carbon (MNC) was estimated as the sum of FNC and BNC.

[0060] Table 2: Statistical results of microbial residue carbon and plant residue carbon Table 3: Statistical results of fungal residue carbon and bacterial residue carbon From Table 2 and Table 3, and Figure 1 and Figure 2 As shown, under NPS3 of the present invention, it can be observed that more bacteria are mobilized, thereby resulting in more and faster conversion of plant residue carbon to microbial residue carbon.

[0061] Table 4: Introduction of carbon, nitrogen and phosphorus acquisition enzymes studied in this patent Table 5: Basic chemical properties of the surface and subsoil at different sampling periods.

[0062] Different lowercase letters in horizontal rows indicate significant differences (P < 0.05) between NPS0 to NPS3 in the top and bottom soils, respectively. Asterisks indicate significant differences (P < 0.05) between the top and bottom soils. SOC, soil organic carbon; NO3--N / NH4+-N, nitrate nitrogen / ammonium nitrogen; SAP, soil available phosphorus; CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added.

[0063] Table 6: Stoichiometric ratios of carbon, nitrogen and phosphorus for enzyme activity.

[0064] Different lowercase letters in vertical columns indicate significant differences between NPS0 to NPS3, and different uppercase letters in horizontal rows indicate significant differences between different sampling times (P < 0.05). BG, β-glucosidase; CBH, cellobiohydrolase; LAP, L-leucyl aminopeptidase; NAG, β-N-acetylglucosaminidase; AP, acid phosphatase. CK, soil only; NPS0, soil with straw only; NPS1 to NPS3, soil with straw and increasing nutrients added.

[0065] The determination method of plant residue carbon of the present invention is as follows: A. Determination of PNC of plant residue carbon Ligninphenol was used as a biomarker. Ligninphenol includes vanillylphenol V, eugenol S and cinnamylphenol C. 0.5 g of air-dried sample was placed in a Monad reactor, and 500 mg of copper oxide, 100 mg of ammonium sulfate, 50 mg of glucose, 0.4 ml of internal standard solution and 15 ml of sodium hydroxide were added; B. The reactor was oxidized at 150°C for 150 minutes; C. Centrifuge the oxidation product (3000 rpm, 15 min), transfer the supernatant to another centrifuge tube (pH: 1.8-2.2), and store in the dark for 1 hour. After centrifugation again, transfer the supernatant to a volumetric flask, titrate to 100 ml with ethyl acetate solution, and evaporate in a rotary evaporator (<38 °C). Dissolve the residue with 0.5 ml of internal standard solution, transfer to a 3 ml derivatization bottle, and dry with N2 gas. D. The dried residue was dissolved with 50 μl pyridine and 100 μl N,O-bis(trimethylsilyl)trifluoroacetamide, derivatized at 60°C for 10 min, and detected by gas chromatography; The PNC of plant residue carbon is calculated according to formula (3): PNC = (C V / 0.33 + C S / 0.9 + C C ) / (0.08 × 1000) (3) Among them, C V , C S and C C are the carbon concentrations in vanillylphenol V, eugenol S, and cinnamylphenol C (ug g –1 ), the coefficients of CuO oxidation to release V, S and C are 0.33, 0.90 and 1 respectively; The minimum lignin concentration in major crop plant residues was set at 0.08; 1000 is the conversion factor between ug and mg.

[0066] Regarding the response of MNC and PNC to substrate stoichiometry, the accumulation of MNC and PNC was affected by the substrate stoichiometry ( Figure 1 ). Compared with NPS0, NPS3 increased the MNC of the topsoil (90th and 150th days) and deep soil by 7.6%–8.4% and 11.8%–18.1%, respectively. Compared with NPS0, NPS3 decreased the PNC of the topsoil and deep soil by 4.4%–17.5% and 10.6%–21.7%, respectively. NPS1 had no significant effect on both MNC and PNC. Under the same treatment, the MNC or PNC of the topsoil was higher than that of the deep soil. Over time, the contribution of MNC to SOC increased from 52.0% to 66.4% in the topsoil and from 42.9% to 53.2% in the deep soil. The contribution of PNC to SOC decreased from 38.9% to 15.3% in the topsoil and from 39.2% to 13.3% in the deep soil.

[0067] FNCs are more sensitive to substrate stoichiometry than BNCs ( Figure 2 ). Compared with NPS0, NPS3 increased the FNC of the topsoil and deep soil by an average of 14.8% and 20.8%, respectively. In contrast, NPS3 reduced the BNC of the topsoil by 6.0%–13.6% (no change in the deep soil). NPS1 had no significant effect on MNC and PNC. The FNC or BNC of the topsoil was 1.4–1.7 times or 0.9–1.3 times that of the deep soil. With the increase in nutrient matching, the intensity of microbial transformation of phenols gradually increased, and the intensity of the deep soil was higher than that of the topsoil ( Figure 3 ).

[0068] Regarding the response of basic soil properties to substrate stoichiometry, NPS1–NPS3 increased the moisture content of deep soil by 8.7%–23.6% on day 30 compared with NPS0, while the surface soil showed no change ( Fig. 9 ). AN and SAP concentrations increased from NPS1 to NPS3. For example, from NPS0 to NPS3, the SAP concentration of the topsoil increased from 18.2 to 32.7 mg kg –1 The activities of BG (NPS3) and AP (NPS0–NPS3) in the deep soil were higher than those in the surface soil at the 30th and 90th days ( Figure 4 ). No changes in LAP activity were observed between NPS0–NPS3. Only NPS3 reduced the Ace of bacteria in the deep soil by 10.6% on day 30 compared with NPS0. No changes were observed in either the surface or deep soil in the other treatments ( Fig.11 a, b, and c). No changes in fungal Ace were observed between NPS0–NPS3 ( Fig.11 g, h, and i).

[0069] In the surface soil, Proteobacteria, Acidobacteria, and Actinobacteria were dominant, accounting for 34%–44%, 17%–22%, and 18%–24%, respectively. In the deep soil, the dominant bacteria were Actinobacteria, Proteobacteria, and Firmicutes, accounting for 24%–42%, 14%–41%, and 8%–19%, respectively ( Figure 5 ). Compared with NPS0, NPS1–NPS3 enriched the abundance of Proteobacteria, Planctomyces, and Bacteroidetes in both the surface and deep soils, while the abundance of Firmicutes increased only in the deep soil. The fungal composition between the surface and deep soils was similar. Compared with NPS0, NPS1 increased the abundance of Morcellaria by 61% and had no effect on the abundance of Ascomycota. NPS3 increased the abundance of Morcellaria and Ascomycota by 23% and –24.7%, respectively ( Fig.12 ).

[0070] Regarding the different regulatory mechanisms of MNC and PNC formation in the surface and deep soil layers, Figure 6 Seventeen variables were used to reflect the effects of soil physical, chemical and biological parameters on NC. For NPS1, Planctomyces, AN, SM, bacterial acetylesterase activity and AP were the most important factors regulating the changes in MNC, and the effects of Mortierellomycota, Firmicutes and SAP on MNC in deep soil increased. For NPS3, the changes in MNC were more dependent on Proteobacteria, AN, SAP, SM and BG, and the effects of bacterial acetylesterase activity on MNC in deep soil increased. The changes in PNC were more dependent on Planctomyces, AN, fungal acetylesterase activity, BG and Mortierella, and the effects of SAP were enhanced in deep soil.

[0071] A schematic diagram of NC evolution was constructed using specific important factors under OM and EM ( Figure 7 ). For NPS1, the dominant bacteria (Planthomycetes, etc.) and their abundance, AP, Nu, and SM controlled the changes in NC. MNC and PNC were more affected by AN and SAP, respectively. Firmicutes had a significant effect on NC in deep soil. For NPS3, more bacteria such as Proteobacteria were mobilized, and BG and AN played an important role in the changes in NC. Fungi and their abundance controlled more in the changes in PNC in the topsoil. From NPS1 to NPS3, the average ratio of MNC to SOC increased from 59% to 62%, while the ratio of PNC decreased from 27% to 22%. It can be seen that under NPS3 of the present invention, more bacteria can be observed to be mobilized, resulting in more and faster conversion of plant residue carbon to microbial residue carbon.

Claims

1. An application method for promoting stable soil carbon deposition based on microbial metabolism, characterized in that: By adding straw carbon and nutrients nitrogen, phosphorus and sulfur, the C:N:P:S stoichiometric ratio of the substrate in the soil was adjusted to 10000:(1034-2004):(167-494):(133-264).

2. The application method of promoting stable soil carbon deposition based on microbial stoichiometric metabolism according to claim 1 is characterized in that the stoichiometric ratio of C:N:P:S is 10000:2004:494:

264.

3. A method for determining soil carbon accumulation based on microbial metabolic activity, characterized in that: The steps include: S1. Establish a control group NPS0, and treatment groups NPS1-NPS3 with combined addition of straw carbon and nutrients nitrogen, phosphorus and sulfur. The stoichiometric ratio of C:N:P:S added to the soil in each treatment of NPS1-NPS3 was adjusted to 10000:(1034-2004):(167-494):(133-264); S2. Soil samples were collected from the topsoil and bottomsoil, air-dried, sieved and impurities removed, and then placed in mesh bags according to the treatment groups NPS0-NPS3. Nine replicates were maintained for each treatment of the topsoil and bottomsoil; S3. Three uniform and regular pits were dug in the field as landfill areas. Three replicates of the four treatments were buried in each pit at different depths of the topsoil and bottomsoil, and the corresponding layers of soil were used for backfilling; S4. Sow corn in the landfill area and regularly take duplicate samples of each treatment from the landfill area to determine the content of microbial residue carbon and plant residue carbon.

4. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: The treatment group NPS0 was the traditional straw return method, and the amount of nitrogen, phosphorus, and sulfur added to NPS0 was 0, which served as the control group; The C:N:P:S stoichiometric ratio of NPS1 is 10000:1034:167:133 to match the metabolic requirements of fungi; The metabolic demand matched by the C:N:P:S stoichiometric ratio of NPS2 is between that of fungi and bacteria, with a stoichiometric ratio of 10000:1556:238:171; The C:N:P:S stoichiometric ratio of NPS3 is 10000:2004:494:264 to match the metabolic requirements of bacteria.

5. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: In step S1, the C added incrementally in the treatment groups NPS0-NPS3 is straw carbon, which is obtained by crushing straw into 2-5 mm fragments, adding 2 g of fragments to every 100 g of soil sample after air drying, sieving and impurity removal, and the concentrations of C, N, P and S in the fragments are 41.76%, 0.974%, 0.120% and 0.119%, respectively; the stoichiometric ratio of the substrate is adjusted by adding nutrient solutions NS1, NS2 or NS3 containing ammonium nitrate, potassium dihydrogen phosphate and ammonium sulfate to the treatment groups NPS1-NPS3, and no nutrient solution is added to the treatment group NPS0, and the concentrations of N, P and S in NS1 are 6.421, 1.791 and 0.952 g L -1 , the concentrations in NS2 were 19.502, 3.521, and 1.902 g L -1 , and the concentrations in NS3 were 30.721, 9.981, and 4.232 g L -1 , the amount of nutrient solution NS1, NS2, and NS3 added was 1 ml per 100 g of soil sample.

6. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: The topsoil is soil with a depth of 0-20 cm, and the bottomsoil is soil with a depth of 20-40 cm. Before the soil substrate samples are treated with the treatment groups NPS0-NPS3, the moisture content of the soil samples is adjusted to 60% of the field water holding capacity with distilled water. The mesh bag used is a nylon mesh bag with a mesh bag pore size of 0.048 mm.

7. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: The pit dimensions are 1.5 m long, 0.5 m wide, 0.4 m deep, and 0.6 m apart. Each treatment is spaced 20 cm apart. The placement depth of each treatment in the bottom soil is 20-40 cm, and the placement depth of each treatment in the top soil is 0-20 cm.

8. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: The corn was planted with a spacing of 27 cm between plants and 60 cm between rows. All repeated samples from a landfill area were collected for measurement at 30, 90 and 150 days after sowing.

9. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: The determination method of the microbial residue carbon is as follows: a. Glucosamine GlcN and muramic acid MurA were used as biomarkers for evaluation. One gram of air-dried sample was hydrolyzed in a hydrolysis tube at 105°C for 8 hours. b. Add 100 μL of inositol to the tube, mix, filter and spin dry at 65°C; c. The residue was dissolved in 20 mL of pure water, centrifuged, and freeze-dried. The freeze-dried residue was dissolved in 5 mL of anhydrous methanol, centrifuged, transferred to a 5 mL derivatization bottle, and dried with N2 gas; d. Add 1 mL of pure water and 100 μL of N-methylglucosamine and freeze-dry; e. Prepare three standard samples, add 100 μL of mixed standard solution to each sample and freeze-dry; f. The gas-dried samples and standard samples were sealed with 300 μL of derivatization reagent and shaken for 30 seconds; g. In a water bath, add 1 mL of acetic anhydride, 1.5 mL of dichloromethane and 1 mL of hydrochloric acid to each sample in sequence; h. After standing and stratification, the inorganic phase was removed and the above operation was repeated 3 times; the remaining organic phase was dried with N2 gas at 45 ° C; the residue was dissolved with 300 μL of diluent and detected by gas chromatography; i. Calculate the bacterial residue carbon BNC and fungal residue carbon FNC according to formulas (1) and (2): BNC = C MurA × 45 (1) FNC = (C GlcN / 179.17 – 2 × C MurA / 251.23) × 179.17 × 9 (2) Among them, C MurA is the concentration of MurA (mg g –1 ), 45 is the conversion factor between MurA and BNC, C GlcN is the concentration of GlcN (mg g –1 ), 179.17 and 251.23 are the relative molecular weights of GlcN and MurA, respectively, and 9 is the conversion coefficient between GlcN and FNC; Microbial residue carbon (MNC) was estimated as the sum of FNC and BNC.

10. The method for determining the promotion of soil stable carbon deposition based on microbial metabolic activity according to claim 3, characterized in that: The determination method of the plant residue carbon is as follows: A. Determination of PNC of plant residue carbon Ligninphenol was used as a biomarker. Ligninphenol includes vanillylphenol V, eugenol S and cinnamylphenol C. 0.5 g of air-dried sample was placed in a Monad reactor, and 500 mg of copper oxide, 100 mg of ammonium sulfate, 50 mg of glucose, 0.4 ml of internal standard solution and 15 ml of sodium hydroxide were added; B. The reactor was oxidized at 150°C for 150 minutes; C. The oxidation product was centrifuged, and the supernatant was transferred to another centrifuge tube and stored in the dark for 1 hour. After centrifugation again, the supernatant was transferred to a volumetric flask, titrated to 100 ml with ethyl acetate solution, and evaporated in a rotary evaporator. The residue was dissolved with 0.5 ml of internal standard solution, transferred to a 3 ml derivatization bottle, and dried with N2 gas; D. The dried residue was dissolved with 50 μl of pyridine and 100 μl of N,O-bis(trimethylsilyl)trifluoroacetamide, derivatized at 60°C for 10 min, and detected by gas chromatography; The PNC of plant residue carbon is calculated according to formula (3): PNC = (C V / 0.33 + C S / 0.9 + C C ) / (0.08 × 1000) (3) Among them, C V , C S and C C are the carbon concentrations in vanillylphenol V, eugenol S, and cinnamylphenol C (ug g –1 ), the coefficients of CuO oxidation to release V, S, and C are 0.33, 0.90, and 1, respectively; The minimum lignin concentration in major crop plant residues was set at 0.08; 1000 is the conversion factor between ug and mg.

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